Previous studies by other investigators have led to the conclusion that methotrexate (MTX) enters mammalian cells via a transport system which also actively transports MTX out of cells and that the resistance of one type of MTX-resistant cell mutant is due to a defect in this transport system. Some experimental observations, however, are inconsistent with these hypotheses and our own preliminary results suggest a different interpretation of reported results. In studies on azaguanine uptake we have observed that the drug is not a substrate for the hypoxanthine/guanine transport system, but instead enters by a non-saturable process, most likely non-mediated permeation and that it enters the cells only in its non-ionized form (pKa equals 6.6). Thus the extent of its intracellular accumulation in cells, in which it is not phosphorylated, depends on both the extracellular and intracellular pH. Intracellular:extracellular steady-state concentration ratios of 3 to 0.1 were observed experimentally. The ionized forms of azaguanine and of other drugs do not permeate the membrane because of their low solubility in membrane lipids. We postulate that the reported results for MTX uptake can be accounted for on the same basis since it has pKas between 3.5 and 5.5. We propose to re-evaluate the mechanism of permeation of MTX through the membranes of wild type and MTX-resistant lines of cultured cells. We will apply improved methodology recently developed by us which allows substrate uptake measurements in time intervals as short as 1.5 sec and yields accurate intracellular water space estimates. MTX uptake studies are complicated by its rapid intracellular binding to dihydrofolate reductase. We will attempt to isolate a mutant cell line deficient in this enzyme which would allow us to clearly distiguish between MTX uptake and intracellular binding.